Monthly Archives: September 2013

3 Top Factors Researchers Consider When Selecting an NGS Provider

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At Genohub, not only do we seek feedback from researchers, our development methodology is almost entirely based on this feedback. We receive this feedback via website forms as well as routine one-on-one conversations with some of the top researchers using next generation sequencing for their projects. Through this data and interaction, certain trends have begun to emerge which may be useful to an NGS provider seeking additional projects. This list is not based on a controlled experiment, however countless conversations indicate that these factors are extremely important:

  1. Turnaround time – this one is a toss up when compared with price, but we typically find turnaround time to be among the leading factors in a researcher’s decision to select an NGS provider. We have heard quite a few stories of researchers seeing turnaround times over several months for library prep and sequencing.
  2. Price – while this is one of the biggest factors for researchers, it must be qualified with established trust which is the next major factor.
  3. Trust – this one is a biggie for many researchers and often a non-starter if not established. The main reasons for this are that researchers are hesitant to ship their precious samples (ie human brain tissue) to an NGS provider for quite often costly sequencing if they are not confident in their abilities. Researchers have told us some of the things they look for which lend to building their confidence:
    • Referrals & Reviews – researchers seek out colleagues who have done similar projects and look for recommendations. Word of mouth is one of the biggest methods researchers rely on to select an NGS provider.
    • Publications – providers who are listed in publications involving similar projects.
    • What kind of QC will be run on the sample.
    • Overall experience indicators such as time in business and volume of samples regularly handled.
    • Data and sample security.
    • Location – this factor is considerably important if previous trust is not established. Some researchers have absolutely no problem shipping samples across the globe, while others might physically drive their samples to a local provider to ensure sample integrity.

We would love to hear your feedback on this topic whether you are an NGS provider, or a researcher actively using next sequencing. What other decision driving criteria have you found as a provider, or what are some other factors important to you as a researcher?

In a Nutshell: Life Tech Exome Certified Service Provider Program

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Life Technologies announced yesterday that they launched the Ion AmpliSeq Exome Certified Service Provider Program.

What the program is in a nutshell:

  • Goals: Offer a network of next gen sequencing providers able to help researchers get a high quality exome sequence at a reduced cost with fast turnaround times and low amounts of input material
  • Exome sequencing inputs: as little as 50ng of customer DNA
  • Library kit used: Ion AmpliSeq Exome kit
  • NGS Instrument used: Ion Proton
  • Exome sequencing outputs: high quality data, which of course can be used with Ion Reporter Software for mutation validation, annotation, and reporting

The Service Provider Program is intended to fill exome sequencing market demand which Life Tech argues has been under-serviced with exome sequencing currently going for $1,000+ , long turnaround times up to 8 weeks, and requiring up to 3mg of DNA. Dr. Candace Johnson, Deputy Director and the Wallace Chair of Translational Research at Roswell Park Cancer Institute states “Exome sequencing will be central to discoveries made in clinical research”. If the Exome CSP delivers as promised, it could have a major impact in accelerating discoveries made in clinical research.

For more information on the Life Tech Provider Program please see the entire press release.

Human Virome Variation

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Bacteriophages, (a virus that infects and replicates within bacteria) are the most abundant biological entity on earth with an estimated population of ∼10^31 particles (Weinbauer, 2004).  Humans are also colonized by an immense population of viruses whose identities vary greatly from person to person. Many of the members of gut viromes are bacteriophages that infect bacterial species found in humans that harbor that particular bacteria. A new study by Minot et al., 2013 demonstrates that in addition to the variability introduced by different bacterial species in the gut, viral populations undergo rapid evolution giving rise to new bacteriophage species. Minot et al., follow an adult male for 2.5 years and examine his viral community by extreme deep sequencing (56 billion bases of purified viral sequence from 24 longitudinal fecal samples). Purified viral DNA was linearly amplified using Φ29 DNA polymerase which showed that bacterial 16S sequences were reduced to less than 10 copies per nanogram of DNA. Paired end reads were then obtained using Illumina HiSeq sequencing yielding more than 573 million reads, with 15-39 million reads per sample. Data analysis identified 478 contigs that were assumed to be DNA viruses. While 60 of these contgs assemble into circles, 87% showed no overlap with previously identified viruses.Taxonomic analysis of these contigs indicated recovery of Microviridae, Podoviridae, Myoviridae, and Siphoviridae, but contigs with taxonomic attributions were a minority, only 13%, emphasizing the enormous sequence variation present in bacteriophages. Interestingly, no human viruses were recovered, suggesting that the gut virome of healthy humans consists of mostly bacteriophages.

So while one factor for viral differences between populations is related to difference in bacterial populations, the authors point out that rapid within-host viral evolution is another contributing factor. Their data suggests that multiple new viral species will commonly arise in the gut of a typical human over the course of a human life.

Minot, S. et al. Rapid evolution of the human gut virome. Proc. Natl Acad. Sci. USA http://dx.doi.org/10.1073/ pnas.1300833110 (2013)

 

Weinbauer MG, Review Ecology of prokaryotic viruses, FEMS Microbiol Rev. May; 28(2):127-81 (2004)

8 Top Online Calculators for Next Gen Sequencing

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When it comes to next gen sequencing, here are the top 8 online calculators that we have found:

Coverage

  1. Genohub’s shop by project feature
  2. The Coverage Calculator by Clinical Genomics

Oligonucleotide

  1. The Oligo Calc: Oligonucleotide Properties Calculator by Northwestern University
  2. The OligoAnalyzer 3.1 by Integrated DNA Technologies

Primers

  1. NCBI/ Primer-BLAST: Finding primers specific to your PCR template (using Primer3 and BLAST) 
  2. Primer3: WWW primer tool by UmassMed

qPCR Efficiency

  1. The qPCR efficiency calculator by Thermo Scientific
  2. The QPCR Standard Curve Slope to Efficiency Calculator by Agilent Technologies

If you know of any other great online calculators for next gen sequencing please tell us about it!